The present invention relates to the use of pharmaceutical agents (compounds) which are known as xcex1hd vxcex23 integrin antagonists in combination with chemotherapeutic agents and methods for using the same for treatment or prevention of neoplasia diseases.
Integrins are a group of cell surface glycoproteins which mediate cell adhesion and therefore are useful mediators of cell adhesion interactions which occur during various biological processes. Integrins are heterodimers composed of noncovalently linked xcex1 and xcex2 polypeptide subunits. Currently eleven different xcex1 subunits have been identified and six different xcex2 subunits have been identified. The various a subunits can combine with various xcex2 subunits to form distinct integrins.
The integrin identified as xcex1vxcex23 (also known as the vitronectin receptor) has been identified as an integrin associated with endothelial cells and smooth muscle cells. xcex1vxcex23 integrins can promote the formation of blood vessels (angiogensis) in tumors. These vessels nourish the tumors and provide access routes into the bloodstream for metastatic cells.
The xcex1vxcex23 integrin is known to play a role in various conditions or disease states including tumor metastasis, solid tumor growth (neoplasia), osteoporosis, Paget""s disease, humoral hypercalcemia of malignancy, angiogenesis, including tumor angiogenesis, retinopathy, including macular degeneration, arthritis, including rheumatoid arthritis, periodontal disease, psoriasis and smooth muscle cell migration (e.g. restenosis). Thus, compounds which selectively inhibit or antagonize xcex1vxcex23 would be beneficial for treating such conditions.
It has been shown that the xcex1vxcex23 integrin and other xcex1v containing integrins bind to a number of Arg-Gly-Asp (RGD) containing matrix macromolecules. Compounds containing the RGD sequence mimic extracellular matrix ligands so as to bind to cell surface receptors. However, it is also known that RGD peptides in general are non-selective for RGD dependent integrins. For example, most RGD peptides which bind to xcex1vxcex23 also bind to xcex1vxcex25, xcex1vxcex21 and xcex1IIbxcex23. Antagonism of platelet xcex1IIbxcex23 (also known as the fibrinogen receptor) is known to block platelet aggregation in humans. In order to avoid bleeding side-effects when treating the conditions or disease states associated with the integrin xcex1vxcex23, it would be beneficial to develop compounds which are selective antagonists of xcex1vxcex23 as opposed to xcex1IIbxcex23.
Tumor cell invasion occurs by a three step process: 1) tumor cell attachment to extracellular matrix; 2) proteolytic dissolution of the matrix; and 3) movement of the cells through the dissolved barrier. This process can occur repeatedly and can result in metastases at sites distant from the original tumor.
Seftor et al. (Proc. Natl. Acad. Sci. USA, Vol. 89 (1992) 1557-1561) have shown that the xcex1vxcex23 integrin has a biological function in melanoma cell invasion. Montgomery et al., (Proc. Natl. Acad. Sci. USA, Vol. 91 (1994) 8856-60) have demonstrated that the integrin xcex1vxcex23 expressed on human melanoma cells promotes a survival signal, protecting the cells from apoptosis. Mediation of the tumor cell metastatic pathway by interference with the xcex1vxcex23 integrin cell adhesion receptor to impede tumor metastasis would be beneficial.
Brooks et al. (Cell, Vol. 79 (1994) 1157-1164) have demonstrated that antagonists of xcex1vxcex23 provide a therapeutic approach for the treatment of neoplasia (inhibition of solid tumor growth) since systemic administration of xcex1vxcex23 antagonists causes dramatic regression of various histologically distinct human tumors.
The adhesion receptor integrin xcex1vxcex23 was identified as a marker of angiogenic blood vessels in chick and man and therefore such receptor plays a critical role in angiogenesis or neovascularization. Angiogenesis is characterized by the invasion, migration and proliferation of smooth muscle and endothelial cells. Antagonists of xcex1vxcex23 inhibit this process by selectively promoting apoptosis of cells in neovasculature. Therefore, xcex1vxcex23 antagonists would be useful therapeutic targets for treating such conditions associated with neovascularization (Brooks et al., Science, Vol. 264, (1994), 569-571).
A neoplasm or tumor, is an abnormal, unregulated, and disorganized proliferation of cell growth. A neoplasm is malignant, or cancerous, if it has properties of destructive growth, invasiveness and metastasis. Invasiveness refers to the local spread of a neoplasm by infiltration or destruction of surrounding tissue, typically breaking through the basal laminas that define the boundaries of the tissues, thereby often entering the body""s circulatory system. Metastasis typically refers to the dissemination of tumor cells by lymphotics or blood vessels. Metastasis also refers to the migration of tumor cells by direct extension through serous cavities, or subarachnoid or other spaces. Through the process of metastasis, tumor cell migration to other areas of the body establishes neoplasms in areas away from the site of initial appearance.
Cancer is now the second leading cause of death in the United States. However, cancer is not fully understood at the molecular level. It is known that exposure of a cell to a carcinogen leads to DNA alteration that inactivates a suppressive gene or activates an oncogene.
Suppressive genes are growth regulatory genes, which upon mutation, can no longer control cell growth. Oncogenes are initially normal genes (called prooncogenes) that by mutation or altered context of expression become transforming genes. The products of transforming genes cause inappropriate cell growth. More than twenty different normal cellular genes can become oncogenes by genetic alteration. Transformed cells differ from normal cells in many ways, including cell morphology, cell-to-cell interactions, membrane content, cytoskeletal structure, protein secretion, gene expression and mortality (transformed cells can grow indefinitely).
Cancer is now primarily treated with one or a combination of three types of therapies: surgery, radiation and chemotherapy. Surgery involves the bulk removal of diseased tissue. While surgery is sometimes effective in removing tumors located at certain sites, for example, in the breast, colon and skin, it cannot be used in the treatment of tumors located in other areas, such as the backbone, nor in the treatment of disseminated neoplastic conditions such as leukemia.
Chemotherapy involves the disruption of all replication or cell metabolism. It is used most often in the treatment of breast, lung and testicular cancer. Many adverse effects are experienced by patients undergoing systemic chemotherapy for treatment of neoplastic diseases. Chemotherapy-induced side effects significantly impact the quality of life of the patient and may dramatically influence patient compliance with treatment.
The present invention relates to the use of compounds of the following general formula 
wherein X and Y are the same or different halo group; R is H or alkyl; and pharmaceutically acceptable salts thereof in combination with chemotherapeutic agents and methods for using the combinations for treatment or prevention of neoplasia diseases.
The compounds described above can exist in various isomeric forms and all such isomeric forms are meant to be included. Tautomeric forms are also included as well as pharmaceutically acceptable salts of such isomers and tautomers.
The present invention relates to a class of compounds known as xcex1vxcex23 integrin antagonists represented by the following formulae I-XVIII, in combination with chemotherapeutic agents, more fully described below, and methods of using such combinations for treatment or prevention of neoplasia diseases. 
wherein R is H or lower alkyl and pharmaceutically acceptable salts thereof.
Among chemotherapuetic agents that may be used in combination with the xcex1vxcex23 antagonist compounds include, but are not limited to, 5-fluorouacil, cyclophosphamide, cisplatin, taxol and doxorubicin are preferred. Other chemotherapeutics useful in combination and within the scope of the present invention include, but are not limited to, buserelin, topoisomerase inhibitors such as topotecan and irinotecan, mitoxantrone, BCNU, CPT-11, chlorotranisene, chromic phosphate, gemcitabine, dexamethasone, estradiol, estradiol valerate, estrogens conjugated and esterified, estrone, ethinyl estradiol, floxuridine, goserelin, hydroxyurea, carboplatin, melphalan, methotrexate, mitomycin and prednisone. Table I lists other chemotherapeutic agents which can be used in the present invention.
Table I provides known median dosages for selected chemotherapeutic agents which may be useful in combination with the xcex1vxcex23 antagonist compounds and compositions.
The following is a list of definitions of various terms used herein:
As used herein, the terms xe2x80x9calkylxe2x80x9d or xe2x80x9clower alkylxe2x80x9d refer to a straight chain or branched chain hydrocarbon radicals having from about 1 to about 10 carbon atoms, and more preferably 1 to about 6 carbon atoms. Examples of such alkyl radicals are methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, pentyl, neopentyl, hexyl, isohexyl, and the like.
As used herein the term xe2x80x9chaloxe2x80x9d or xe2x80x9chalogenxe2x80x9d refers to bromo, chloro, or iodo.
As used herein the term xe2x80x9chaloalkylxe2x80x9d refers to alkyl groups as defined above substituted with one or more of the same or different halo groups at one or more carbon atom. Examples of haloalkyl groups include trifluoromethyl, dichloroethyl, fluoropropyl and the like.
The term xe2x80x9ccompositionxe2x80x9d as used herein means a product which results from the mixing or combining of more than one element or ingredient.
The term xe2x80x9cpharmaceutically acceptable carrierxe2x80x9d, as used herein means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting a chemical agent.
The term xe2x80x9ctherapeutically effective amountxe2x80x9d shall mean that amount of drug or pharmaceutical agent that will elicit the biological or medical response of a tissue, system or animal that is being sought by a researcher or clinician.
The following is a list of abbreviations and the corresponding meanings as used interchangeably herein:
The compounds described herein can exist in various isomeric forms and all such isomeric forms are meant to be included. Tautomeric forms are also included as well as pharmaceutically acceptable salts of such isomers and tautomers.
In the structures and formulas herein, a bond drawn across a bond of a ring can be to any available atom on the ring.
The term xe2x80x9cpharmaceutically acceptable saltxe2x80x9d refers to a salt prepared by contacting a compound described above with an acid whose anion is generally considered suitable for human consumption. Examples of pharmacologically acceptable salts include the hydrochloride, hydrobromide, hydroiodide, sulfate, phosphate, acetate, propionate, lactate, maleate, malate, succinate, tartrate salts and the like. All of the pharmacologically acceptable salts may be prepared by conventional means. (See Berge et al., J Pharm. Sci., 66(1), 1-19 (1977) for additional examples of pharmaceutically acceptable salts.)
Treatment or prevention of a neoplasia disease in a mammal is provided by methods and combinations using one or more xcex1vxcex23 integrin antagonist described above with one or more chemotherapeutic agents described above. The method comprises treating a mammal with a therapeutically effective amount of an xcex1vxcex23 integrin antagonist in combination with a chemotherapeutic agent.
xcex1vxcex23 inhibitors are being developed as potential anti-cancer agents. Compounds that impair endothelial cell adhesion via the xcex1vxcex23 integrin induce improperly proliferating endothelial cells to die.
The xcex1vxcex23 integrin has been shown to play a role in melanoma cell invasion (Seftor et al., Proc. Natl. Acad. Sci. USA, 89: 1557-1561, 1992). The xcex1vxcex23 integrin expressed on human melanoma cells has also been shown to promote a survival signal, protecting the cells from apoptosis (Montgomery et al., Proc. Natl. Acad. Sci., USA, 91: 8856-8860, 1994).
Mediation of the tumor cell metastatic pathway by interference with the xcex1vxcex23 integrin cell adhesion receptor to impede tumor metastasis would be beneficial. Antagonists of xcex1vxcex23 have been shown to provide a therapeutic approach for the treatment of neoplasia (inhibition of solid tumor growth) because systemic administration of xcex1vxcex23 antagonists causes dramatic regression of various histologically distinct human tumors (Brooks et al., Cell, 79: 1157-11164, 1994).
The adhesion receptor identified as integrin xcex1vxcex23 is a marker of angiogenic blood vessels in chick and man. This receptor plays a critical role in angiogenesis or neovascularization. Angiogenesis is characterized by the invasion, migration and proliferation of smooth muscle and endothelial cells by new blood vessels. Antagonists of xcex1vxcex23 inhibit this process by selectively promoting apoptosis of cells in the neovasculature. The growth of new blood vessels also contributes to pathological conditions such as diabetic retinopathy (Adamis et al., Amer. J. Opthal., 118: 445-450,1994) and rheumatoid arthritis (Peacock et al., J. Exp. Med., 175:, 1135-1138, 1992). Therefore, xcex1vxcex23 antagonists can be useful therapeutic targets for treating such conditions associated with neovascularization (Brooks et al., Science, 164: 569-571, 1994).
There are five major classes of chemotherapeutic agents currently in use for the treatment of cancer: natural products and their derivatives; anthracyclins; alkylating agents; antimetabolites; and hormonal agents. Chemotherapeutic agents are often referred to as antineoplastic agents.
The alkylating agents are believed to act by alkylating and cross-linking guanine and possibly other bases in DNA, arresting cell division. Typical alkylating agents include nitrogen mustards, ethyleneimine compounds, alkyl sulfates, cisplatin, and various nitrosoureas. A disadvantage with these compounds is that they not only attack malignant cells, but also other cells which are naturally dividing, such as those of bone marrow, skin, gastrointestinal mucosa and fetal tissue.
Antimetabolites are typically reversible or irreversible enzyme inhibitors, or compounds that otherwise interfere with the replication, translation or transcription of nucleic acids.
Several synthetic nucleosides have been identified that exhibit anticancer activity. A well known nucleoside derivative with strong anticancer activity is 5-fluorouacil. 5-Fluorouracil has been used clinically in the treatment of malignant tumors, including, for example, carcinomas, sarcomas, skin cancer, cancer of the digestive organs, and breast cancer. 5-Fluorouacil, however, causes serious adverse reactions such as nausea, alopecia, diarrhea, stomatitis, leukocytic thrombocytopenia, anorexia, pigmentation and edema.
Cytosine arabinoside (also referred to as Cytarabin, araC, and Cytosar) is a nucleoside analog of deoxycytidine that was first synthesized in 1950 and introduced into clinical medicine in 1963. It is currently an important drug in the treatment of acute myeloid leukemia. It is also active against acute lymphocytic leukemia, and to a lesser extent, is useful in chronic myelocytic leukemia and non-Hodgkin""s lymphoma.
The following table provides illustrative examples of median dosages for selected cancer agents that may be used in combination with an xcex1vxcex23 integrin antagonist agent. It should be noted that the specific dose regimen for the chemotherapeutic agents below will depend upon dosing considerations based upon a variety of factors including the type of neoplasia; the state of the neoplasm; the age, weight, sex, and medical condition of the patient; the route of administration; the renal and hepatic function of the patient; and the particular combination employed.
The compounds of the formula I-XVIII are potent and selective, orally available, small molecule peptidomimetic antagonists of xcex1vxcex23. These compounds were designed to explore the utility of xcex1vxcex23 antagonists in preclinical models of angiogensis and solid tumor growth. XII was found to be a potent (IC50 less than 1 nM) and selective (IC50=0.2xcexcM vs xcex1IIbxcex23) antagonist of xcex1vxcex23 in vitro. Human microvessel endothelial cell proliferation and migration was found to be dependent on xcex1vxcex23 and XII dose-dependently inhibited these functions. In vivo, XII was a potent inhibitor of angiogenesis. Oral administration of XII (40 mg/kg) significantly inhibited angiogenesis in the mouse corneal micropocket assay. Moreover, XII was a potent inhibitor of solid tumor growth in vivo. XII dose-dependently inhibited M21 human melanoma tumor growth in SCID mice over a dose range of 0.2-30 mg/kg. Inhibition of tumor growth with XII was additive to that using the chemotherapeutic agent, cisplatin, at a maximum tolerated dose. Together, these results suggest that the xcex1vxcex23 antagonists described herein will be effective therapeutic agents against the growth of solid tumors in the clinic.
Implantation of Rice Leydig tumor cells subcutaneously in the flank of SCID mice led to the growth of a large tumor (volume greater than 1500 mm3) within 11 days and the development of severe hypercalcemia ( greater than 15 mg/dl). The xcex1vxcex23 antagonist XII administered orally, inhibited tumor growth and hypercalcemia in a dose-dependent manner. Cisplatin treatment (maximum tolerated dose) of tumor bearing mice inhibited tumor growth by approximately 50%. XII (10 mg/kg, PO) alone inhibited growth by 10%, but in combined therapy with cisplatin, tumor growth was reduced 80% compared to control-treated mice. Survival in this model is a function of hypercalcemia as well as tumor growth. Cisplatin or XII alone had little effect on survival time. However, the combined cisplatin and XII treatment almost doubled overall survival. These results clearly demonstrate the efficacy of the orally administered xcex1vxcex23 antagonist, XII, to reduce the growth of a solid tumor and associated hypercalcemia when used as monotherapy. Moreover, XII in combination with the chemotherapeutic agent, cisplatin, was shown to have superior efficacy to either agent alone. XII and other similar xcex1vxcex23 antagonists should provide important therapeutic opportunities to treat cancer.
The methods and combination therapy of the present invention provide one or more benefits. Combinations of xcex1vxcex23 integrin antagonists with chemotherapeutic agents are useful in treating and preventing neoplasia diseases. Preferably, the xcex1vxcex23 integrin antagonist agent or agents and the chemotherapeutic compounds, compositions, agents and therapies of the present invention are administered in combination at a low dose, that is, at a dose lower than has been conventionally used in clinical situations for each of the individual components administered alone.
A benefit of lowering the dose of the compounds, compositions, agents and therapies of the present invention administered to a mammal includes a decrease in the incidence of adverse effects associated with higher dosages. For example, by lowering the dosage of a chemotherapeutic agent such as methotrexate, a reduction in the frequency and the severity of nausea and vomiting will result when compared to that observed at higher dosages. Similar benefits are contemplated for the compounds, compositions, agents and therapies in combination with the xcex1vxcex23 integrin antagonist agents of the present invention.
By lowering the incidence of adverse effects, an improvement in the quality of life of a patient undergoing treatment for cancer is contemplated. Further benefits of lowering the incidence of adverse effects include an improvement in patient compliance, a reduction in the number of hospitalizations needed for the treatment of adverse effects, and a reduction in the administration of analgesic agents needed to treat pain associated with the adverse effects.
When administered as a combination, the therapeutic agents can be formulated as separate compositions which are given at the same time or different times, or the therapeutic agents can be given as a single composition.
When used as a therapeutic agent the compounds described herein are preferably administered with a physiologically acceptable carrier. A physiologically acceptable carrier is a formulation to which the compound can be added to dissolve it or otherwise facilitate its administration. Examples of physiologically acceptable carriers include, but are not limited to water, saline, physiologically buffered saline. Additional examples are provided below.
The xcex1vxcex23 integrin antagonist compounds of the present invention may be administered orally, parenterally, or by inhalation spray, or topically in unit dosage formulations containing conventional pharmaceutically acceptable carriers, adjuvants and vehicles. The term parenteral as used herein includes, for example, subcutaneous, intravenous, intramuscular, intrasternal, infusion techniques or intraperitonally.
The xcex1vxcex23 integrin antagonist compounds of the present invention are administered by any suitable route in the form of a pharmaceutical composition adapted to such a route, and in a dose effective for the treatment intended. Therapeutically effective doses of the compounds required to prevent or arrest the progress of or to treat the medical condition are readily ascertained by one of ordinary skill in the art using preclinical and clinical approaches familiar in the medicinal arts.
The present invention provides a method of treating or preventing neoplasia diseases which method comprises administering a therapeutically effective amount of a compound selected from the class of xcex1vxcex23 integrin antagonist compounds described above, in combination with a chemotherapeutic agent, wherein one or more compound is administered in association with one or more non-toxic, pharmaceutically acceptable carrier and/or diluent and/or adjuvant (collectively referred to herein as xe2x80x9ccarrierxe2x80x9d materials) and if desired other active ingredients.
Based upon standard laboratory experimental techniques and procedures well known and appreciated by those skilled in the art, as well as comparisons with compounds of known usefulness, the compounds described above can be used in the treatment of patients suffering from neoplasia diseases. One skilled in the art will recognize that selection of the most appropriate xcex1vxcex23 integrin antagonist+chemotherapeutic compound of the invention is within the ability of one with ordinary skill in the art and will depend on a variety of factors including assessment of results obtained in standard assay and animal models.
Treatment of a patient afflicted with neoplasia disease comprises administering to such a patient an amount of xcex1vxcex23 integrin antagonist compound in combination with a chemotherapeutic agent described above which is therapeutically effective in controlling the condition or in prolonging the survivability of the patient beyond that expected in the absence of such treatment. As used herein, the term xe2x80x9cinhibitionxe2x80x9d of the condition refers to slowing, interrupting, arresting or stopping the condition and does not necessarily indicate a total elimination of the condition. It is believed that prolonging the survivability of a patient, beyond being a significant advantageous effect in and of itself, also indicates that the condition is beneficially controlled to some extent.
The dosage regimen for the compounds and/or compositions containing the compounds is based on a variety of factors, including the type, age, weight, sex and medical condition of the patient; the severity of the condition; the type of neoplasia disease; the route of administration; and the activity of the particular compound employed. Thus the dosage regimen may vary widely. Dosage levels of the order from about 0.01 mg to about 1000 mg per kilogram of body weight per day of the xcex1vxcex23 integrin antagonist compounds are useful in the treatment of the neoplasia diseases, and more preferably from about 0.01 mg to about 100 mg per kg of body weight per day. The amount that can be combined with the chemotherapeutic agent to produce a single dosage form will vary depending upon the host treated and the particular mode of administration.
The active xcex1vxcex23 integrin antagonist ingredient administered by injection is formulated as a composition wherein, for example, saline, dextrose or water may be used as a suitable carrier. A suitable daily dose would typically be about 0.01 to 10 mg/kg body weight injected per day in multiple doses depending on the factors listed above.
For administration to a mammal in need of such treatment, the compounds in a therapeutically effective amount are ordinarily combined with one or more adjuvants appropriate to the indicated route of administration. The compounds may be admixed with lactose, sucrose, starch powder, cellulose esters of alkanoic acids, cellulose alkyl esters, talc, stearic acid, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric and sulphuric acids, gelatin, acacia, sodium alginate, polyvinylpyrrolidone, andlor polyvinyl alcohol, and tableted or encapsulated for convenient administration. Alternatively, the compounds may be dissolved in water, polyethylene glycol, propylene glycol, ethanol, corn oil, cottonseed oil, peanut oil, sesame oil, benzyl alcohol, sodium chloride, and/or various buffers. Other adjuvants and modes of administration are well and widely known in the pharmaceutical art.
It is understood that a specific dose level for any particular patient will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, rate of excretion, drug combination, and the severity of the particular neoplasia disease being treated and form of administration.
Treatment dosages generally may be titrated to optimize safety and efficacy. Typically, dosage-effect relationships from an initial in vitro analysis can provide useful guidance on the proper doses for patient administration. Studies in animal models also generally may be used for guidance regarding effective dosages for treatment of cancers in accordance with the present invention. In terms of treatment protocols, it should be appreciated that the dosage to be administered will depend on several factors, including the particular agent that is administered, the route administered, the condition of the particular patient, etc. It will generally be desirable to administer the xcex1vxcex23 integrin antagonist agents either orally, parenterally, intravenously, or subcutaneously. Other routes of administraton are also contemplated, including intranasal and transdermal routes, and by inhalation. Generally speaking, one will desire to administer an amount of the agent that is effective to achieve a serum level commensurate with the concentrations found to be effective in vitro. Thus, where an agent is found to demonstrate in vitro activity at, e.g., 10 xcexcM, one will desire to administer an amount of the drug that is effective to provide about a 10 xcexcM concentration in vivo. Determination of these parameters are well within the skill of the art. These considerations, as well as effective formulations and administration procedures are well known in the art and are described in standard textbooks.
Any effective treatment regiment can be utilized and readily determined and repeated as necessary to effect treatment. In clinical practice, the compositions containing the xcex1vxcex23 integrin antagonist alone or in combination with other therapeutic agents can be administered in specific cycles until a response is obtained.
For patients who are without advanced or metastatic cancer, an xcex1vxcex23 integrin antagonist based drug in combination with one or more anticancer agents can be administered as an immediate initial therapy prior to surgery, chemotherapy, or radiation therapy, and as a continuous post-treatment therapy in patients at risk for recurrence or metastasis. The goal in these patients is to inhibit the growth of potentially metastatic cells from the primary tumor during surgery or radiotherapy and inhibit the growth of tumor cells from undetectable residual primary tumor.
For patients who are with advanced or metastatic cancer, an xcex1vxcex23 integrin antagonist based drug in combination with one or more anticancer agents of the present invention can be used as a continuous supplement to, or possible replacement for, hormonal ablation. The goal in these patients is to slow or prevent tumor cell growth from both the untreated primary tumor and from the existing metastatic lesions.
In addition, the invention may be particularly efficacious during post-surgical recovery, where the present compositions and methods may be particularly effective in lessening the chances of recurrence of a tumor engendered by shed cells that cannot be removed by surgical intervention.
The xcex1vxcex23 integrin antagonist may be used in conjunction with one or more other treatment modalities, including, but not limited to surgery and radiation, hormonal therapy, immunotherapy, and cryotherapy. The present invention can be used in conjunction with any current or future therapy.
The phrase xe2x80x9ccombination therapyxe2x80x9d (or xe2x80x9cco-therapyxe2x80x9d), in defining the use of an xcex1vxcex23 integrin antagonist compound and chemotherapeutic agent or therapy of the present invention, is intended to embrace administration of each agent or therapy in a sequential manner in a regimen that will provide beneficial effects of the combination, and is intended as well to embrace co-administration of these agents or therapies in a substantially simultaneous manner, such as in a single capsule having a fixed ratio of these active agents or in multiple, separate capsules for each agent.
The term xe2x80x9ctreatmentxe2x80x9d refers to any process, action, application, therapy, or the like, wherein a mammal, including a human being, is subject to medical aid with the objective of improving the mammal""s condition, directly or indirectly.
The term xe2x80x9cinhibitionxe2x80x9d, in the context of neoplasia, tumor growth or tumor cell growth, may be assessed by delayed appearance of primary or secondary tumors, slowed development of primary or secondary tumors, decreased occurrence of primary or secondary tumors, slowed or decreased severity of secondary effects of disease, arrested tumor growth and regression of tumors amongst others. In the extreme, complete inhibition, is referred to herein as prevention.
The term xe2x80x9cprevention,xe2x80x9d in relation to neoplasia, tumor growth or tumor cell growth, means no tumor or tumor cell growth if none has occurred, and no further tumor or tumor cell growth if there had already been tumor growth.
The term xe2x80x9cangiogenesisxe2x80x9d refers to the process by which tumor cells trigger abnormal blood vessel growth to create their own blood supply, and is a major target of cancer research. Angiogenesis is believed to be the mechanism via which tumors get needed nutrients to grow and metastasize to other locations in the body. Antiangiogenic agents interfere with these processes and destroy or control tumors.
Angiogenesis is an attractive therapeutic target because it is a multistep process that occurs in a specific sequence, thus providing several possible targets for drug action. Antiangiogenic therapy may offer several advantages over conventional chemotherapy for the treatment of cancer.
The xcex1vxcex23 integrin antagonist agents have low toxicity in preclinical trials and development of drug resistance has not been observed (Folkman, J., Seminars in Medicine of the Beth Israel Hospital, Boston 333(26): 1757-1763, 1995). As angiogenesis is a complex process made up of many steps including invasion, proliferation and migration of endothelial cells, combination therapies will be effective in inhibiting angiogensis.
The phrase xe2x80x9ctherapeutically-effectivexe2x80x9d is intended to qualify the amount of each agent that will achieve the goal of improvement in neoplastic disease severity and the frequency of incidence over treatment of each agent by itself, while avoiding adverse side effects typically associated with alternative therapies.
A xe2x80x9ctherapeutic effectxe2x80x9d relieves to some extent one or more of the symptoms of a neoplasia disease. In reference to the treatment of a cancer, a therapeutic effect refers to one or more of the following: 1) reduction in the number of cancer cells; 2) reduction in tumor size; 3) inhibition (i.e., slowing to some extent, preferably stopping) of cancer cell infiltration into peripheral organs; 4) inhibition, to some extent, of tumor growth; 5) relieving or reducing to some extent one or more of the symptoms associated with the disease; and/or 6) relieving or reducing the side effects associated with the administration of anticancer agents.
xe2x80x9cTherapeutic effective amountxe2x80x9d is intended to qualify the amount required to relieve to some extent one or more of the symptoms of a neoplasia disease. In reference to the treatment of a cancer, a therapeutic effect refers to one or more of the following: 1) reduction in the number of cancer cells; 2) reduction in tumor size; 3) inhibition (i.e., slowing to some extent, preferably stopping) of cancer cell infiltration into peripheral organs; 4) inhibition (i.e., slowing to some extent, preferably stopping) of tumor metastasis; 5) inhibition, to some extent, of tumor growth; 6) relieving or reducing to some extent one or more of the symptoms associated with the disorder; and/or 7) relieving or reducing the side effects associated with the administration of anticancer agents.
The pharmaceutical compositions useful in the present invention may be subjected to conventional pharmaceutical operations such as sterilization and/or may contain conventional pharmaceutical adjuvants such as preservatives, stabilizers, wetting agents, emulsifiers, buffers, etc.
The general synthetic sequences for preparing the xcex1vxcex23 integrin antagonist compounds useful in the present invention are outlined in Schemes I-III. Both an explanation of, and the actual procedures for, the various aspects of the present invention are described where appropriate. The following Schemes and Examples are intended to be merely illustrative of the present invention, and not limiting thereof in either scope or spirit. Those with skill in the art will readily understand that known variations of the conditions and processes described in the Schemes and Examples can be used to synthesize the xcex1vxcex23 integrin antagonist compounds of the present invention.
Unless otherwise indicated all starting materials and equipment employed were commercially available. 
Scheme I illustrates methodology useful for preparing the tetrahydropyrimidinobenzoic acid portion of the xcex1vxcex23 integrin antagonist which can be coupled to a gly-xcex2-amino acid ester. Briefly, in Scheme I, 3,5-dihydroxybenzoic acid is converted to 3-amino-5-hydroxy-benzoic acid using the procedure described in Austr. J. Chem., 34 (6), 1319-24 (1981). The product is reacted with ammonium thiocyanate in hot dilute hydrochloric acid to give 3-thiourea-5-hydroxybenzoic acid after normal work-up. This thiourea intermediate is converted to the S-methyl derivative by reaction with methyl iodide in ethanol at reflux. 1,3-diamino-2-hydroxypropane is reacted with this resulting intermediate in hot DMA. Upon cooling precipitate forms and the zwitterionic product is isolated by filtration. The HCl salt may be obtained by lyophilizing from dilute hydrochloric acid. Alternatively, the product may be isolated from the original reaction mixture by removing volatiles and concentrating. The resulting product is taken up in water and pH adjusted to about 5-7 where zwitterionic product precipitates and is isolated by filtration. The HCl salt may be obtained as previously stated or by simply dissolving in dilute hydrochloric acid and concentrating to a solid and drying. 
Scheme IA illustrates methodology useful for preparing the tetrahydropyrimidinobenzoic acid portion of the xcex1vxcex23 integrin antagonist which can be coupled to a gly-xcex2-amino acid ester. Briefly, in Scheme IA 1,3-diamino-2-hydroxypropane is reacted with carbon disulfide in an appropriate solvent such as ethanolxe2x80x94water, refluxed, cooled, hydrochloric acid added, refluxed again, cooled and the product, 5-hydroxytetrahydropyrimidine-2-thione harvested by filtration and dried. This cyclic thiourea intermediate is converted to the S-methyl derivative by reaction of thione and methyl iodide in ethanol at reflux. The desired 2-methylthioether-5-hydroxypyrimidine hydroiodide is readily isolated by removing volatiles at reduced pressure. Thus, 2-methylthioether-5-hydroxypyrimidine hydroiodide in methylene chloride: DMA (about 10:1) and an equivalent of triethylamnine are cooled to about ice-bath temperature and an equivalent of di-tert-butyl dicarbonate (BOC anhydride) added. Conventional work-up gives the BOC-2-methylthioether-5-hydroxypyrimidine as an oil.
3,5-dihydroxybenzoic acid is converted to 3-amino-5-hydroxybenzoic acid using the procedure of Aust. J. Chem., 34 (6), 1319-24 (1981).
The final desired product, 3-hydroxy-5-[(5-hydroxy-1,4,5,6-tetrahydro-2-pyrimidinyl)amino]benzoic acid hydrochloride salt, is prepared by reacting BOC-2-methylthioether-5-hydroxypyrimidine and 3-amino-5-hydroxy-benzoic acid in hot DMA. Upon cooling, a precipitate forms and zwitterionic product isolated by filtration. The HCl salt can be obtained by lyophilizing from dilute hydrochloric acid, for example. 
Scheme II illustrates methodology useful for preparing the ethyl N-gly-amino-3-(3,5-dihalo-2-hydroxy)phenyl propionate portion of the xcex1vxcex23 integrin antagonist which can be coupled to the tetrahydropyrimidinobenzoic acid moiety. Briefly, 3,5-halo substituted salicylaldehydes may be prepared by direct halogenation as, for example, would be the case where 5-bromosalicylaldehyde is slurried in acetic acid and an equivalent or more of chlorine is added to yield 3-chloro-5-bromo-2-hydroxybenzaldehyde. Some product precipitates and can be recovered by filtration. The remainder may be recovered by diluting the filtrate with water and isolating the precipitate. Combining the solids and drying gives 3-chloro-5-bromo-2-hydroxybenzaldehyde. 3-iodo-5-chlorosalicylaldehyde may be prepared by reacting 5-chlorosalicylaldehyde with N-iodosuccinimide in DMF and subjecting the reaction mixture to usual work-up conditions. 3-iodo-5-bromosalicylaldehyde may be prepared by reacting 5-bromosalicylaldehyde in acetonitrile with potassium iodide and chloramine T. Work-up gives a material that when treated with hexanes gives the desired 3-iodo-5-chlorosalicylaldehyde.
Coumarins are readily prepared from salicylaldehydes using a modified Perkin reaction (e.g., Vogel""s Textbook of Practical Organic Chemistry, 5th Ed., 1989, p. 1040,). The halo-substituted coumarins are converted to 3-aminohydrocoumarins (see J. G. Rico, Tett. Let., 1994, 35, 6599-6602) which are readily opened in acidic alcohol to give 3-amino-3-(3,5-halo-2-hydroxy)phenyl propanoic acid esters.
3-amino-3-(3,5-halo-2-hydroxy)phenyl propanoic acid esters are converted to N-gly-3-amino-3-(3,5-halo-2-hydroxy)phenyl propanoic acid esters by reaction of Boc-N-gly-N-hydroxysuccinimide to give Boc-N-gly-3-amino-3-(3,5-halo-2-hydroxy)phenyl propanoic acid esters that are converted to HX salts of N-gly-3-amino-3-(3,5-halo-2-hydroxy)phenyl propanoic acid esters (wherein X is a halo group) for example, by removal of the BOC-protecting group using HCl in ethanol.
The amino acid compounds used in preparing the xcex1vxcex23 integrin antagonist compounds of the present invention can be prepared according to the procedures set forth herein and below and according to the methodology described and claimed in co-pending U.S. Ser. No. 60/076,710 filed Mar. 4, 1998. 
Scheme III is illustrative of methodology useful for preparing various xcex1vxcex23 integrin antagonist compounds of the present invention. 3-Hydroxy-5-[(1,4,5,6-tetrahydro-5-hydroxy-2-pyrimidinyl)amino]benzoic acid is activated to coupling using known methods. Thus, after dissolving in a suitable solvent such as DMA an equivalent of NMM is added. The reaction mixture is cooled to ice-bath temperatures and IBCF added. To the mixed anhydride intermediate is added the gly-xcex2-amino acid ester and NMM. Upon completion of the reaction the product is purified by prep hplc and the ester hydrolyzed to the acid by treating with a base, such as LiOH in a suitable solvent (dioxane/water or acetonitrile/water). Alternatively, a suitable acid, such as TFA can be used. The product is isolated by prep hplc or by isolating the zwitterion at pH 5-7 and converting to the desired salt by standard procedures.